Biofilm

Staphylococcus aureus is a leading cause of biofilm-associated infections, in which communities of bacterial cells are encased in an extracellular matrix composed of polysaccharides, proteins, and extracellular DNA (eDNA) that protect bacteria from host immune defense and antibiotics. Despite their importance, the mechanisms by which matrix components are released from bacterial cells and incorporated into the biofilm matrix remain poorly understood. Using a drip-flow biofilm system, we showed that MVs were associated with the biofilm matrix formed by S. aureus clinical isolate MN8. Proteomic analysis of biofilm matrix proteins and purified MVs showed that biofilm-derived MVs carried cytoplasmic, membrane, and extracellular proteins that closely resembled the protein composition of the biofilm matrix but differed significantly from MVs produced by planktonic cultures. Biofilm-derived MVs carried significantly higher levels of DNA than MVs from planktonic cultures, and MV-associated DNA was resistant to DNase treatment. Although strain MN8 is known to form polysaccharide-dependent biofilms, exogenously added DNase or proteinase K significantly impaired biofilm formation and integrity. Notably, these inhibitory effects were reversed by the addition of biofilm-derived MVs, which significantly restored biofilm formation in enzyme-treated cultures. Together, these findings provide evidence that S. aureus MVs are generated within biofilms, and that these MVs serve as an important resource of matrix components and contribute to biofilm formation. ImportanceExtracellular membrane vesicles (MVs) are important mediators of intercellular communication and have been implicated in the physiology and pathogenesis of bacterial infections. While MV production in S. aureus planktonic cultures has been recognized for over one decade, their presence and function in S. aureus biofilm formation have remained unexplored. Here, we report for the first time the purification and characterization of MVs derived from S. aureus biofilms. Our studies demonstrate that S. aureus MVs are important components of the biofilm matrix that contribute to biofilm formation by serving as key carriers of matrix proteins and eDNA. This work advances our limited understanding of MVs in Gram-positive bacteria and reveal a previously unrecognized mechanism underlying S. aureus biofilm formation.

Rock-inhabiting fungi thrive in subaerial oligotrophic environments such as desert rocks, solar panels and marble monuments where organic carbon and nitrogen are scarce. We tested whether the rock-inhabiting fungus Knufia petricola showed a preference regarding nitrogen ([Formula] or [Formula]) and carbon (glucose or sucrose) sources and whether it was sensitive towards carbon and nitrogen limitation. As this fungus produces the carbon-rich, nitrogen-free 1,8-dihydroxynaphthalene (DHN) melanin, we tested whether a melanin-deficient mutant would be less sensitive to carbon limitation. The carbon and nitrogen concentrations were the primary predictors of growth, with a broad optimum partially explained by an optimal fungal C:N ratio. Limiting carbon or nitrogen supply decreased biomass formation, CO2 production and biofilm thickness but promoted substratum penetration through filamentous growth. The nitrogen content of the biomass was flexible within limits, increasing upon increasing nitrogen supply or decreasing carbon supply. The carbon use efficiency was fairly constant, whereas melanization correlated with a higher nitrogen content of the biomass despite melanin being nitrogen-free. In conclusion, in vitro, K. petricola switches to explorative growth under nutrient limitations, like fast-growing fungi, revealing universal fungal resource-acquisition patterns. Graphical abstract text and imageCarbon and nitrogen availability affect biofilm growth and morphology of the extremotolerant fungus Knufia petricola Abolfazl Dehkohneh, Julia Schumacher, Bastiaan J. R. Cockx, Karin Keil, Tessa Camenzind, Jan-Ulrich Kreft, Anna A. Gorbushina, Ruben Gerrits Growth of the rock-inhabiting fungus Knufia petricola was studied by varying carbon and nitrogen sources and concentrations. Overall, growth was best predicted by the carbon and nitrogen concentrations. Carbon and nitrogen limitation promoted substratum penetration through filamentous growth. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/712823v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@6d98bdorg.highwire.dtl.DTLVardef@146aac5org.highwire.dtl.DTLVardef@757fa8org.highwire.dtl.DTLVardef@ff709_HPS_FORMAT_FIGEXP M_FIG C_FIG

Biofilm formation is a major cause of chronic implant-related bone infections and is associated with impaired immune responses. In a previous study, we identified the cGAS-STING pathway as a potential therapeutic target, as its activation--observed in response to planktonic Staphylococcus aureus (SA)--was absent in the corresponding biofilm setting. The present study aimed to identify potential mechanisms underlying the lack of cGAS activation in the biofilm environment. As biofilm-derived nucleases might degrade cGAS ligands, we assessed presence and activity of micrococcal nuclease in conditioned media from planktonic and biofilm-grown SA and evaluated the impact of extracellular DNases on cGAS pathway activation in macrophages. In addition, we examined altered cGAS expression, the requirement for continuous biofilm exposure and potential downstream inhibition resulting from degradation of the cGAS product. Biofilm formation was associated with dynamic nuclease expression, and exposure to the biofilm environment led to reduced cGAS levels in macrophages, accompanied by a lack of interferon response. Exogenous cGAS activation by G3-YSD failed to restore signaling, independent of nuclease activity or continuous biofilm exposure. In contrast, supplementation with the cGAS product and STING ligand 2'3'-cGAMP fully restored interferon responses and enhanced macrophage activation, indicating that increased degradation of the second messenger in the biofilm environment is not responsible for impaired pathway activation. Similar effects observed with Staphylococcus epidermidis and primary macrophages suggest a broader mechanism that is not SA- or cell line-specific. In conclusion, our data provide novel mechanistic insight into biofilm-mediated impairment of cGAS-STING signaling, revealing a previously unrecognized mechanism of immune evasion in staphylococcal biofilms. These findings extend our previous work and support the therapeutic potential of targeting STING as promising strategy to restore immune responses in chronic implant-related bone infections. HighlightsO_LIBiofilm-derived factors impair cGAS-STING pathway activation and suppress interferon responses in macrophages. C_LIO_LIImpaired signaling is not primarily explained by extracellular micrococcal nuclease-mediated degradation of potential cGAS ligands. C_LIO_LIBiofilm exposure reduces cGAS expression levels and inhibits exogenous cGAS activation independently of continuous presence. C_LIO_LIExogenous 2'3'-cGAMP fully restores interferon responses, indicating that impaired signaling is not due to degradation of the cGAS product. C_LIO_LIDirect activation of STING broadly enhances macrophage activation and by this could amplify overall immune responses. C_LIO_LIBypassing cGAS via direct STING targeting represents a potential therapeutic strategy to overcome immune evasion in chronic implant-related bone infections. C_LI

BackgroundThe nasal microbiome is a collection of diverse microbial populations that inhabit the nose. Staphylococcus aureus is the most common opportunistic pathogen that colonizes the nasal mucosa, increasing the risk of invasive infections in immunocompromised and hospitalized patients. Clinicians usually prescribe antibiotics to decolonize the nasal cavities of at-risk patients from S. aureus. However, their broad antimicrobial activity can damage the resident nasal microbiome. Instead, naturally occurring compounds or resident bacteria in nasal microbiomes can effectively and safely exclude S. aureus from the nose. Cell culture and animal models have been used for nasal microbiome studies. However, their unstable microbiomes reduce the accuracy and reliability of the results. Recently, continuous bioreactors have been proposed as alternatives to these models. ResultsWe designed and operated a continuous bioreactor system to maintain stable nasal microbiomes. Next, we inoculated the bioreactor with nasal-swab specimens that we had collected from healthy volunteers. We operated the bioreactors under varying conditions (i.e., operating mode, dilution rate, temperature, pH, and medium composition), and determined the optimal conditions (continuous mode, 1 d-1, 30xlink:href=" pH 6.5, and synthetic nasal medium 3), resulting in stable microbiomes consisting of the main nasal bacterial species. The nasal microbiomes in the optimized bioreactors showed high reproducibility and resilience during a pH perturbation. Moreover, all microbiomes in the bioreactor, which were inoculated with six different nasal-swab specimens, maintained stable bacterial and metabolite compositions. In addition, we applied a synthetic microbial community (SynCom), which was derived from one of the volunteers, to demonstrate a S. aureus decolonization strategy. The bioreactor, inoculated with this SynCom, maintained a stable nasal microbiome for more than one month. Finally, different S. aureus strains that we inoculated in the SynCom showed distinct growth patterns within the otherwise stable community. ConclusionsThe continuous bioreactor enables the cultivation of stable nasal microbiomes for longer than one month by mimicking the environmental conditions of the human nose. The bioreactor is a valuable model for understanding the functions of the nasal microbiome and devising new decolonization strategies against S. aureus.

Unicellular microorganisms can make a transition to multicellular states that enhance survival under environmental fluctuations. In bacteria, one of these states is the biofilm, defined by the production of an extracellular matrix. Although biofilm maturation and dispersion have been extensively studied, where and how initial matrix production is induced within a growing population remains largely unknown. Here we show that production of colanic acid, an important matrix component, is initiated around topological defects, where cell orientation mismatches and growth-induced pressure builds up, in bacterial monolayers. Using Escherichia coli reporting mechanically induced production of colanic acid in response to cell contact and deformation, we found matrix production accompanied by out-of-plane growth under agar-pad confinement. Controlling confinement geometry using microfluidic devices dictated the positions of topological defects and thereby localized regions of high matrix production. These findings reveal that the cell orientation patterning spatially organizes mechanical cues to induce matrix production for biofilm initiation of bacteria.

ObjectivesMultidrug-resistant (MDR) Klebsiella pneumoniae is an increasingly important cause of recurrent urinary tract infections (UTIs), particularly in high-risk patients such as those with neurogenic bladder, where therapeutic options are limited. Bacteriophage therapy represents a promising alternative, but pre-clinical models and characterization of phages active against UTI-derived strains remain scarce. We therefore aimed to isolate and characterize bacteriophages targeting a clinical MDR K. pneumoniae strain causing recurrent UTI and evaluate their activity under urinary conditions. MethodsThree bacteriophages were isolated from environmental samples using an ESBL-producing K. pneumoniae clinical isolate obtained from a neurogenic bladder patient. Phages were characterized by genome sequencing, electron microscopy, stability assays, one-step growth curves, and host-range analysis across 79 clinical UTI isolates. Phage activity was quantified in LB medium and human urine using bacterial growth kinetics and a lytic activity score. ResultsThree lytic phages from the former siphoviridae family (EDIRA083, EDIRA088, and EDIRA092) belonging to distinct genera were identified. Genomic analysis confirmed the absence of lysogeny-associated, virulence, or antibiotic-resistance genes. Latent periods ranged from 8 to 40 minutes and burst sizes from 38 to 170 virions per infected bacterium. Host-range analysis revealed narrow activity for EDIRA083 and EDIRA088, whereas EDIRA092 infected 29% of the 79 clinical isolates tested. In liquid phage infection assays, overall lytic activity was consistently higher and more sustained in human urine than in LB, suggesting reduced fitness of resistant mutants under urinary conditions. ConclusionsThese results identify three genetically distinct lytic phages targeting MDR K. pneumoniae and highlight the importance of testing phage activity under infection-relevant conditions. Their activity in urine supports further evaluation of these phages as candidates for therapeutic development against MDR Klebsiella UTI.

Bacteria exhibit two lifestyles: planktonic free-floating individual cells or sessile multicellular aggregates known as biofilms. The biofilm lifecycle is characterised by three distinct stages: attachment, maturation and dispersal. Distinct adaptations occur in each stage, determining cellular behaviours such as surface attachment or synthesis and degradation of extracellular matrix components. Characterising stage-specific bacterial profiles therefore represents a valuable strategy for the development of novel antibiofilm therapies. Here, we used the model biofilm-forming bacterium Pseudomonas aeruginosa PAO1 to characterise the transcriptional profiles of each stage of the biofilm life cycle: attachment, biofilm maturation and spontaneous dispersal in closed cultures. We report that surface attachment was accompanied by the upregulation of genes comprising the Pil-Chp mechanosensory system, whereas biofilm maturation was characterised by the upregulation of genes involved in Pel polysaccharide synthesis, siaD and PA4396 diguanylate cyclases as well as pipA, fimX and PA5442. In contrast, dispersing cells upregulated genes responsible for the biosynthesis of alginate, rhamnolipid, and extracellular nucleases (eddA, eddB), as well as the transcriptional regulator of dispersal amrZ. Additionally, genes encoding the spontaneous dispersal molecule cis-2-decenoic acid (dspS and dspI), canonical phosphodiesterases (nbdA and rbdA), four non-canonical HD-GYP phosphodiesterases and seven other c-di-GMP-related enzymes were also upregulated during dispersal. Our comprehensive analysis of transcriptional changes across biofilm stages therefore provides benchmarking stage-specific transcriptional profiles for P. aeruginosa biofilms in closed culture systems. Furthermore, it allowed the identification of a subset of fourteen genes as transcriptional biomarkers of dispersal, which were used to build reporter plasmids as tools to determine the onset of dispersal. ImportanceBiofilm infections by P. aeruginosa are a major medical challenge due to the increased tolerance to antimicrobials displayed by bacteria living in sessile communities, which is reduced during spontaneous biofilm dispersal. Attachment, biofilm maturation and dispersal represent the main stages of a dynamic process known as the biofilm lifecycle. However, the global regulatory responses governing transitions between these stages remain understudied. Here, we combine live microscopy and biomass quantification to track the progression of P. aeruginosa cultures through the three main stages of the biofilm lifecycle. We show that cells from each stage recapitulate canonical, stage-specific transcriptional responses and identify a set of biomarkers associated with the onset of dispersal. These biomarkers may offer a practical tool for rapidly screening dispersal-inducing compounds, aiding in the discovery of the next generation of antibiofilm therapeutics.

Bone infections, which are predominantly caused by Staphylococcus (S.) aureus, can be difficult to treat and have high rates of chronicity and reoccurrence. We previously identified that osteoclasts, the cells that break down bone matrix, may contribute to disease progression by allowing S. aureus to replicate intracellularly. There we identified that this bacteriums ability to grow intracellularly is tied to the maturation of osteoclasts. In this study we addressed whether osteoclast differentiation supports intracellular growth by changing the host cells response to infection or by altering the host cell environment to better support S. aureus. Using dual species RNA-sequencing we analyzed host and bacterial transcripts of infected osteoclast and precursor bone marrow macrophage (BMM) cultures. Host transcript analysis suggests that infected osteoclasts are slow to upregulate bacterial response genes compared to BMMs. We also identify that the S. aureus transcriptional response is primarily determined by the host cell type, and that bacteria in osteoclasts upregulate carbon metabolism genes compared to those inside BMMs. By utilizing intracellular survival assays on S. aureus mutants deficient in carbon metabolism and related pathways we determine that S. aureus require glycolysis, acetyl-CoA synthesis, and aspartate biosynthesis for proliferation inside osteoclasts, although bacteria can survive without them. With differentiation, osteoclasts increase glutamine uptake, and this metabolite is required for S. aureus intracellular growth. Taken together, these findings suggest that osteoclasts support S. aureus intracellular growth by providing nutrients required to replicate in the context of a blunted antimicrobial response. IMPORTANCEInfectious osteomyelitis, bone infection, is frequently caused by the bacterium Staphylococcus aureus. Intracellular infection of cells that build bone, osteoblasts, and cells that resorb bone, osteoclasts, have been implicated in disease progression by providing a niche for immune evasion. While S. aureus in osteoblasts are largely quiescent, bacteria in osteoclasts proliferate and therefore may be a source of reemergent infection. Factors that promote this growth in osteoclasts are poorly characterized. In this study we find that osteoclasts have a diminished transcriptional response to infection and show that S. aureus acquire glucose and glutamine, which have high flux in osteoclasts, to support intracellular growth. We further observe that S. aureus in osteoclasts require aspartate synthesis to grow intracellularly. This work highlights the importance of host cellular metabolism for the intracellular fate of S. aureus as an added factor beyond the direct antimicrobial response.

Mycobacteria form rough and smooth colonies. The Mycobacterium marinum strain 1218S is a smooth colony forming variant isolated from the 1218R strain, which forms rough colonies and is more virulent than 1218S in infecting fish. Genes for the type VII secretion ESX-1 system, which includes mycobacterial virulence genes, have been partially duplicated in M. marinum and is refered to as ESX-6. We recently reported that several ESX-1 genes are missing in the 1218S strain. On the basis of the complete genomes of these two and three other M. marinum strains we provide insight into strain differences and similarities focusing on 1218R and 1218S, and ESX genes, selected virulence genes, and LOS genes, which are involved in lipooligosaccharide synthesis and smooth colony formation. We provide RNA-Seq data for 1218R and 1218S and two other well-characterized M. marinum strains suggesting that loss of ESX-1 genes in 1218S results in increased transcript levels of ESX-6 genes. Furthermore, while there is no difference in gene synteny and sequence of LOS genes comparing 1218R and 1218S, with the exception of duplication of lsr2, a regulator of LOS genes, in 1218S. Our RNA-Seq data show increased transcript levels of LOS genes in stationary 1218S cells relative to 1218R indicating that transcription and/or RNA degradation of LOS genes influence smooth and rough colony formation. We finally provide data suggesting that Ms1 RNA affect the transcription of LOS genes (and ESX-1 genes), and that loss of ESX-1 genes influence biofilm formation.

Streptococcus mutans is a primary architect of dental caries, utilizing complex genetic networks to build resilient, acid-producing biofilms. While pooled screens (Tn-seq) have identified important fitness factors, they often fail to capture extracellular or moderate-effect determinants due to community-level masking. Therefore, to study biofilm phenotypes, we constructed a comprehensive arrayed library of 9,216 mutants and used Cartesian Pooling-Coordinate Sequencing (CP-CSeq) to establish a sequence-defined resource covering 51% of non-essential genes. By screening the entire collection in isolation, we identified several novel biofilm determinants, including the putative metal transporter SMU_635 and the glycosylation-associated protein SMU_2160. However, systematic whole-genome sequencing (WGS) of our hits revealed an interesting level of genomic instability: 25% of biofilm-defective mutants had undergone spontaneous recombination at the gtfBC locus, while 7% had lost the TnSmu1 element, an excision rate 1,000-fold higher than previously reported. While targeted mutagenesis confirmed that TnSmu1 loss does not impact biofilm integrity, the gtfBC deletions directly accounted for the most severe phenotypes, highlighting a systemic risk of misattributing gene functions to primary transposon insertions. Our findings provide a powerful new genetic resource for the S. mutans community while establishing a critical new standard: an arrayed library is only as defined as its underlying genome, making systematic genomic verification an essential prerequisite for accurate functional genomics. ImportanceStreptococcus mutans is a major human pathogen responsible for dental caries, a global public health challenge driven in part by the organisms ability to form resilient, acidogenic biofilms. While traditional pooled genetic screens have identified many fitness factors, they often fail to capture extracellular or moderate-effect determinants because neighboring healthy bacteria can mask these defects. This work provides the scientific community with a sequence-defined arrayed mutant library, an essential resource for dissecting the individual contributions of genes to biofilm integrity in isolation. Beyond identifying well-known machinery, this study uncovers novel determinants, including the putative metal transporter SMU_635 and the putative glycosylation-associated protein SMU_2160. Crucially, the discovery of pervasive genomic instability within the library, specifically at the gtfBC and TnSmu1 loci, reveals a systemic risk in functional genomics: the potential to misattribute phenotypes to primary mutations when the underlying background has undergone large-scale rearrangements. By establishing systematic whole-genome verification as a necessary standard, this research ensures that the identification of future therapeutic targets is built upon a verified genetic foundation.

Staphylococcus aureus is the most common bacterial pathogen affecting pediatric patients with Cystic fibrosis (CF), a genetic disorder that causes thick mucus buildup in the lungs, providing a scaffold for chronic infections. Antibiotic treatment is typically guided by standard in vitro antimicrobial susceptibility testing (AST) in Mueller-Hinton broth (MHB), which does not represent the infection site in CF lungs. Notably, discordances between AST predictions and antibiotic therapeutic outcomes were reported in up to 50% of CF cases. To address this gap, we conducted ASTs against methicillin-resistant S. aureus (MRSA) in CF sputum-mimetic media compared with MHB, demonstrating [≥]4-fold discordances across four of nine antibiotic classes. Most significantly, we observed unexpected {beta}-lactam sensitization of MRSA strains (up to 128-fold) in CF sputum-like media, crossing the CLSI clinical breakpoint, suggesting this shift may alter therapeutic outcomes. Genome-wide screens and follow-up assays revealed underlying cell envelope remodelling and alterations to cell envelope stress responses. On the other hand, mucin binding to daptomycin may have led to an apparent 8-fold increase in resistance to this antibiotic in one of the CF sputum-like media. Overall, our AST results in CF sputum-mimetic conditions provide insights into bacterial responses during CF infections. Importantly, they suggest {beta}-lactams may be effective in treating MRSA infections in CF patients, warranting further investigation in relevant in vivo systems.

BackgroundPelagic Sargassum has undergone significant range expansion and dramatic blooms in the Atlantic over the past 15 years. This algaes microbiome provides symbiotic functions that are believed to contribute to its ecological success. Recent research shows that Sargassum-associated bacteria are enriched in integrated prophages compared to the surrounding seawater and that these prophages are inducible by chemical and ultraviolet treatment. ResultsHere, we investigated a Sargassum-derived in vitro multispecies biofilm encompassing the dominant heterotrophic microbial members associated with Sargassum to probe the impacts of prophage induction on the composition of Sargassum microbiomes. Induction was quantified by coverage-based virus-to-host ratios in chemically induced treatments with Mitomycin C and non-induced controls, and the community composition and metabolic profiles were analyzed after a period of recovery post-induction. Chemical induction led to a significant increase in abundance and virus-to-host ratio of viral genomes linked to Vibrio metagenome-assembled genomes. This was accompanied by altered biofilm community composition, with a reduction in Vibrio bacterial abundance that opened niche space for other biofilm members in the genera Pseudoalteromonas, Alteromonas, and Cobetia. The induced Vibrio-associated phages encoded genes involved in quorum sensing, biofilm formation, virulence, and host metabolism. Induction led to a relative loss of 17 metabolic modules, including functions related to energy metabolism and nitrogen utilization. ConclusionDue to the high frequency of lysogeny in the Sargassum microbiome and the susceptibility of prophages to chemical and ultraviolet light induction, these results suggest that prophage integration and induction are mechanisms that significantly contribute to structuring the Sargassum microbiome and its functional profiles, potentially aiding in microbiome flexibility in changing environmental contexts.

The phosphodiesterase (PDE) NbdA (NO-induced biofilm dispersion locus A) consists of a membrane-integrated MHYT domain, a degenerated diguanylate cyclase (DGC) AGDEF domain and an EAL domain. The integral membrane domain MHYT is proposed to sense a so far unknown extracellular signal and transfers the information to the cytosolic enzyme domains to modulate cellular c-di-GMP level. Here, we show that full length NbdA from Pseudomonas aeruginosa PAO1 is an active PDE in vivo. In line with its PDE activity, overexpression leads to slightly reduced global c-di-GMP levels, and reduced twitching motility. Surprisingly, overexpression of truncated cytosolic NbdA variants exhibited increased c-diGMP levels, suggesting previously uncharacterized DGC activity despite lacking a canonical GGDEF motif. While full-length NbdA overexpression resulted in only slight c-di-GMP reduction, cytosolic variants induced a significant increase, indicating a potential for nonenzymatic effects like protein-protein interactions. Further investigation revealed a connection between NbdA and type IV pilus (T4P) function. Overexpression of NbdA conferred resistance to the T4P-dependent phage DMS3vir, suggesting interference with T4P assembly or function. Microscopic analysis demonstrated dynamic localization of NbdA, partially co-localizing with T4P components, supporting a role in T4P regulation. However, no clear link was re-established with flagellar motor switching or chemotaxis signaling. These findings position NbdA in the complex signaling network of c-diGMP and T4P-mediated surface behavior in P. aeruginosa. Future work will focus on elucidating the precise mechanisms of NbdAs PDE activity and its interplay with other DGC/PDE networks. ImportanceIn this work, we show the in vivo activity of the membrane-bound phosphodiesterase NbdA of Pseudomonas aeruginosa, its role in c-di-GMP homeostasis, cellular localization and implications in surface behavior. Using strains overexpressing NbdA and truncated protein variants, we detected a strong defect in growth on solid surfaces and an altered phage susceptibility. Co-localization experiments supported further the hypothesis of interaction with the type IV pilus apparatus. We propose for NbdA to be part of the protein network responsible for c-di-GMP level modulation at the cell pole and thereby regulating the function of type IV pilus apparatus.

Endocavity ultrasound transducers, particularly transvaginal ultrasound (TVUS) probes, contain intricate structures such as notches, grooves, lens surfaces, and handle edges that are highly susceptible to microbial contamination yet difficult to disinfect using conventional high-level disinfection (HLD) methods. This study evaluated the efficacy of a novel ultraviolet-C light-emitting diode (UV-C LED) HLD system in eliminating microbial contamination from these complex probe surfaces. Two TVUS probes were sampled from predefined high-risk regions before and after disinfection following clinical use. Probe A was sampled at the top and bottom notches and both sides of the handle, while Probe B was assessed at the lens, edges, and bent groove regions. Microbial contamination was quantified using swab sampling, culture on agar plates, and identification via MALDI-TOF. Environmental sampling of examination and disinfection rooms was also performed. To assess this system robustness, probe sites were repeatedly inoculated with Bacillus subtilis spores and evaluated following UV-C treatment. Before UV-C treatment, contamination rates ranged from 25% to 57% across sampled regions, with microbial loads reaching up to 3.9 log CFU. Identified organisms included Staphylococcus epidermidis, Pseudomonas koreensis, Bacillus cereus, and Propionibacterium spp. Probe sheaths were also predominantly contaminated with Staphylococcus epidermidis., with counts reaching up to 4.3 log CFU, Environmental sampling revealed diverse microbiota, with higher contamination levels in examination rooms compared to disinfection areas. Following 90 seconds of UV-C exposure, no microbial growth was detected on any sampled site, indicating 100% decontamination. Additionally, UV-C treatment achieved >6.7 log reduction of B. subtilis spores across all tested regions. These findings demonstrate that UV-C LED technology provides rapid, effective, and consistent high-level disinfection of complex TVUS probe surfaces, supporting its potential as a rapid and reliable disinfection modality in clinical setting.

To establish infection, uropathogens must overcome several host defenses including the glycosaminoglycan (GAG) layer coating the apical surface of the bladder urothelium. GAGs are thought to protect against urinary tract infection (UTI) by serving as scaffolding sites for commensals, providing barrier function and preventing uropathogen adherence. However, the ability of uropathogens to degrade and utilize GAGs and the contribution of these activities toward UTI progression is largely unknown. We previously discovered that the uropathogen Proteus mirabilis, a common cause of catheter-associated UTI (CAUTI), degrades the GAG chondroitin sulfate (CS). In this study we sought to define the kinetics and regulation of CS degradation by diverse P. mirabilis strains clinically isolated from both recurrent UTI and CAUTI patients. We found variation in CS degradation kinetics between P. mirabilis strains and media types. However, CS degradation depended on conserved putative chondroitin sulfate ABC endo- and exolyases in all strains. Furthermore, we found that CS degradation in Pm123 was repressed by urea and that this repression was dependent on P. mirabilis urease activity. Complementation of the Pm123 endolyase into urea-insensitive HI4320 resulted in a urea-sensitive CS degradation phenotype suggesting functional differences between the Pm123 and HI4320 endolyases. Sequence alignment and structural modeling analysis identified two unique point mutations within the Pm123 endolyase that may contribute to urea sensitivity. Finally, unlike urea-insensitive P. mirabilis strains, Pm123 demonstrated attenuated swarming and loss of chondroitin endolyase activity had no effect on Pm123 virulence in a mouse CAUTI model. Our results suggest that the kinetics and regulation of CS degradation differ between P. mirabilis strains and in urea-sensitive strains, thus reduces the contribution of CS degradation to urovirulence during murine CAUTI. ImportanceThis work demonstrates that the ability to degrade a common component of bladder mucosal surfaces, chondroitin sulfate, is a phenotype that is shared by multiple strains of the common catheter-associated UTI (CAUTI) pathogen P. mirabilis. We find that this activity is dependent on encoded chondroitin ABC endo- and exolyases, first described in Proteus vulgaris. Additionally, we discovered that for P. mirabilis strain Pm123, degradation of CS is negatively regulated by the presence of urea, a major component of urine. The repression of CS degradation by urea is dependent on the activity of the P. mirabilis urease enzyme, which breaks down urea producing ammonia which raises pH. We found expression of the Pm123 CS endolyase was sufficient to confer a urea-sensitive CS-degradation phenotype and identified two unique mutations within the Pm123 enzyme that may contribute to urea sensitivity. Finally, we find that while CS-degradation plays a role in progression and severity of murine CAUTI model in urea-insensitive P. mirabilis, there was not significant difference in CAUTI outcomes between the urea-sensitive Pm123 wild-type and chondroitinase knockout strains. This study represents a major step forward in understanding the diversity of CS degradation activity and regulation among clinical strains of the critically important CAUTI pathogen P. mirabilis as well as its contribution to urovirulence.

Escherichia coli ST131 is a globally disseminated multidrug-resistant lineage frequently associated with recalcitrant urinary tract infections (UTIs) and bacteraemia. While bacteriophages offer a promising alternative treatment to antibiotics, their efficacy is often limited in physiologically relevant conditions in comparison to laboratory media. In this study, we have investigated the mechanisms by which the representative ST131 strain, EC958, evades elimination by a model phage, LUC4. We observed that in the urine environment, EC958 can transiently resist phage infection by a density dependent mechanism and by the production of protective polysaccharides. Based on this understanding, we developed a phage treatment strategy that can sterilise an EC958 culture in urine-based medium, even at high bacterial densities. The rational design of the successful phage therapy strategy utilises a tailored phage cocktail containing phage that encode depolymerase enzymes to degrade bacterial surface carbohydrates and the targeting of multiple receptors to prevent the emergence of fixed genetic resistant mutants. We found the addition of specific carbon sources renders the bacteria more susceptible to phage infection. By combining these findings with a simulated bladder wash to model voiding, we successfully achieved elimination of EC958 cultures in a urine environment. This study provides a framework for overcoming both fixed and reversible phage resistance, offering a translatable strategy for effectively treating urinary tract infections with phage. Author SummaryIn this study, we investigated how bacterial populations can overcome a phage infection. Phage are viruses that naturally kill bacteria and provide an alternative treatment to antibiotics. We focussed on a particularly aggressive and antibiotic resistant strain of E. coli, EC958, which belongs to a group of E. coli strains that are a leading cause of urinary tract infections and life-threatening bloodstream infections worldwide. We found that in a simulated bladder environment, these bacteria do not rely on genetic mutations to survive but they employ a range of hide and seek strategies. We showed that bacteria can coat themselves in a protective layer to block the phage and use social signalling to enter a dormant state when cell density is high. When they are in this sleep-like state the phage cannot successfully infect. To overcome these bacterial defences, we developed a treatment strategy combining effective phage with specific naturally occurring additives, that trick the bacteria into waking up and becoming vulnerable again to phage infection. By also simulating a clinical bladder wash to reduce bacterial numbers and therefore reduce social-signalling, we were able to eliminate the bacterial population. Our findings suggest that by understanding bacterial strategies we can design more effective and personalised phage therapies to treat bacterial infections.

Standard in vitro antimicrobial susceptibility testing (AST) using Mueller-Hinton broth (MHB) does not reflect infection-site conditions, and its results often do not correlate with therapeutic outcomes. Here, we compared the antibiotic susceptibility of methicillin-resistant Staphylococcus aureus (MRSA), a common chronic wound pathogen, in simulated wound fluid (SWF) resembling wound exudate versus MHB, revealing discordant AST results across six of nine tested antibiotic classes. The most significant were 128-fold increased resistance to tetracyclines and 256-fold sensitization to {beta}-lactams in SWF. Tetracycline resistance was mediated by MntC, an extracellular manganese-binding protein, whereas {beta}-lactam sensitization was driven by cell envelope remodelling in SWF. Galleria mellonella wound infection results matched the SWF susceptibility phenotypes, suggesting SWF better predicts in vivo wound infection therapeutic outcomes. These comprehensive phenotypic and mechanistic insights into MRSA antibiotic responses under wound-infection-mimetic conditions with direct in vivo validation identify a potential new antibiotic adjuvant target and may guide improved antibiotic therapy for MRSA wound infections.

AimsSponge microbiomes have been extensively studied, in part due to their potential as sources of novel antimicrobials and other biologics, with most research focusing on Demosponges. Here, we investigate the Hexactinellid sponge Pheronema carpenteri, previously identified as a promising source of antibiotic-producing bacteria. MethodsUsing next-generation sequencing of bacterial 16S rRNA genes and a single sponge metagenome, we examined the composition of bacterial communities of P. carpenteri sponges recovered from the Porcupine Seabight, along with local water and sediment samples. ResultsOur results show that P. carpenteri harbours a microbiome abundant in Proteobacteria (47.1-59.4%) and Actinobacteria (11.5-27.5%), with consistent intra-aggregation similarities and structured intra-sponge communities. A metagenomic analysis revealed the presence of several nitrogen cycling genes (nirK, nosZ, nirS homologues of proteobacterial origin), supporting a suggestion that these sponges may play a role in nitrogen cycling, while biosynthetic gene clusters (BGCs) were limited (4 complete clusters). Notably, bacterial community structures within P. carpenteri aggregations resemble those observed in both low and high microbial abundance (LMA/HMA) sponges. ConclusionsHexactinellids are traditionally considered LMA sponges, so identifying species that deviate from this dichotomy provides new insights into sponge microbiome ecology. Integrating Hexactinellids into both culture-dependent and culture-independent studies will advance our broader understanding of sponge-associated microbial diversity and could inform biodiscovery programmes in marine environments. Impact StatementOur findings support the suggestion that a combination of culture-based and molecular analyses is required to generate a comprehensive picture of the biosynthetic potential of P. carpenteri sponges. We also reveal insights into the ecosystem services that sponge microbiomes may contribute towards. These observations could facilitate a deeper understanding of the biotechnological and environmental value of key marine resources.

Short-chain fatty acids (SCFAs) like butyrate and propionate are abundant microbiota-derived metabolites that influence bacterial physiology in host-associated niches such as the gastrointestinal tract. However, their effects on Staphylococcus aureus under varying nutritional conditions remain incompletely understood. Here we investigated how SCFAs interact with glucose or galactose to regulate anaerobic growth, biofilm formation, and global transcription in S. aureus. Both SCFAs inhibit growth in a dose-dependent manner. Biofilm formation was differentially affected, with butyrate promoting and propionate suppressing biofilm formation. Glucose and galactose alleviated SCFA-mediated growth inhibition, with glucose exerting the strongest effect. Notably, glucose enhanced butyrate-associated growth and biofilm formation beyond glucose alone, whereas galactose produced more modest effects. Enzymatic and genetic analyses indicated that SCFA-sugar biofilms contain proteins and extracellular DNA and involve VraSR-dependent regulation. Transcriptomic profiling revealed broad metabolic reprogramming, including induction of urease genes, amino acid biosynthesis, and stress response pathways. Synergistic effects between butyrate and glucose were partially dependent on anaplerotic metabolism via pyruvate carboxylase, linking the TCA cycle to SCFA adaptation. Together these findings demonstrate that the nutritional environment dictates whether SCFAs impair S. aureus growth or reprogram its physiology, promoting metabolic adaptation and biofilm formation under sugar-replete conditions.

Streptococcus gallolyticus subsp. gallolyticus (Sgg) is an opportunistic pathobiont associated with bacteremia, infective endocarditis, and colorectal cancer. However, the genomic diversity of this subspecies and the distribution of key virulence determinants, particularly the type VII secretion system (T7SS), remain poorly characterized. Here, we performed genomic analyses of 76 Sgg strains from diverse geographic and host origins. Core- and pan-genome analyses, multi locus sequence typing, and phylogenetic reconstruction revealed dominant sequence types (STs) that correlate with geographic origin or source of isolation. Furthermore, systematic characterization of the T7SS locus identified five new T7SS subtypes and demonstrated a strong association between T7SS subtype and ST. We further expanded the known repertoire of T7SS LXG domain-containing polymorphic toxins (LXG toxins) in Sgg substantially through genome-wide searches. Distinct distribution patterns were observed for the LXG toxins across the strains. Lastly, our data indicated that T7SS subtype was significantly associated with biofilm formation capacity of Sgg strains. Together, these findings advance our understanding of Sgg genomic diversity, reveal substantial lineage-associated variation in T7SS architecture and effector repertoires, and suggest a previously unrecognized connection between T7SS and biofilm formation in Sgg.